Since the advent of well-developed and standard techniques for the syntheses of uniformly .sup.13 C/.sup.15 N-labeled proteins and ribonucleic acids (RNA), multinuclear magnetic resonance (NMR) spectroscopy has been routinely applied for the determination of their structures. By comparison, the determination of DNA structures by multidimensional NMR is infrequent primarily due to the lack of economic and efficient techniques for the synthesis of uniformly .sup.13 C/.sup.15 N-labeled DNA molecules. The availability of .sup.13 C/.sup.15 N-labeling allows resolution and sequential assignment of spin-systems belonging to individual nucleotides in a long DNA duplex which, in turn, results in the generation of a large set of distance constraints that are essential for determining high-resolution structures. The generation of NMR spectra of uniformly labeled DNA enables the delineation of the structures of DNA complexes with specific proteins and the identification of conformational changes in DNA upon protein binding.
In principle, labeled DNA molecules can be chemically or enzymatically synthesized. However, the preparation of such oligodeoxynucleotides (DNA) by chemical synthesis is uncommon because the generation of uniformly .sup.13 C/.sup.15 N-labeled, chemically protected mononucleotides required for phosphoramidite polymerization is costly, inefficient and technically challenging, since such species are not available commercially. DNA labeling by enzymatic methods was introduced by D. P. Zimmer et al. in "NMR Of Enzymatically Synthesized Uniformly .sup.13 C/.sup.15 N-Labeled Oligonucleotides," Proc. Natl. Acad. Sci., U.S.A. 92, 3091 (1995) and includes template-directed synthesis using Klenow DNA polymerase and a mixture of labeled deoxynucleoside triphosphate precursors (dNTPs). However, control of the extent of chain elongation is difficult; therefore, for preparation of labeled DNA having a desired length, hairpin templates must be designed such that the single-stranded overhang in the stem controls the length of the newly synthesized DNA strand. Alternatively, an RNA primer may be used in the enzymatic synthesis of labeled DNA having a defined length. In both procedures, uniformly .sup.13 C/.sup.15 N-labeled DNA duplexes are obtained by adding equimolar quantities of two complementary strands made from two different templates. Inherent in these methods, then, is a step in which the newly synthesized labeled strand is separated from an unlabeled template. This is achieved using denaturing gel electrophoresis, followed be elution of the DNA from the gel matrix. Poor yields of labeled DNA and residual polyacryamide contamination are concerns for NMR applications.
The Polymerase Chain Reaction (PCR) has recently been used to prepare uniformly isotope-labeled DNA oligonucleotides. In "Preparation Of Uniformly Isotope-Labeled DNA Oligonucleotides For NMR Spectroscopy," by John M. Louis et al. J. Bio. Chem. 273, 2374 (1998), the chain extension is based upon self-priming which leads to synthesis of multiple copies of the sequence of interest having flanking restriction sites. Self-priming PCR works well only for GC-rich DNA strands, but not for AT-rich strands, since for AT-rich DNA strands, the melting temperature is significantly lowered and the accuracy of amplification deteriorates. Additionally, Louis et al. reports that some of the oligonucleotides cannot be cleaved to monomers, so that an additional chromatography step is necessary to remove the approximately 10% of unwanted dimeric material present after the DNA amplification.
Accordingly, it is an object of the present invention to provide a general method for preparing uniformly .sup.13 C/.sup.15 N-labeled single- or double-stranded oligodeoxynucleotides with high efficiency and accuracy.
Another object of the present invention is to provide a general method for preparing uniformly .sup.13 C/.sup.15 N-labeled single- or double-stranded oligodeoxynucleotides, without the necessity for complex purification steps.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.